Separations of nitrogenous organic compounds



June 23, 1959 J, STARK 2,891,945

SEPARATIONS OF NITROGENOUS ORGANIC COMPOUNDS Fild July 18, 1955 3Sheets-Sheet 1 MODIFICATION A u b I c MIXTURE TO BE TREATED WASH DO-WATER CONTAINS,FOR EXAMPLEI WATER I ELUANT PURINES, PYRIMIDINES, Nuc-LEOSIDES, AMINO ACIDS.

NEUTRAL ORGANIC. COMPOUNDS, e.g.,$UGARS. INORGANIC SALTS,e.g., KCI,NOC|

NON-AMINO CARBOXYLIC ACIDS, e.g.,LACTlC ACID SOLUTIONS APPLIED TOEXGHANGER SERIALLY IN ORDER: 0 b c.

REMAINING ON EXCHANGER AFTER ELUTIONZ EXCHANGER NON-AMINO CARBOXYLIG :2AOIOs, suc As LACTIC. FORM INORGANIC ANIONS SUCH As OI',so

COLORED PRINCIPLES d e WASH WATER EFFLUENT ELUATE OONTAINs: cONTAINs:

SUGARS AND OTHER NEUTRAL PuRINEs, PYRIMIDINE s, ORGANIC COMPOUNDS.NUGLEOSIDES, AMINO ACIDS INORGANIC cATIONs, SUCH AS NcI K+, etc.

EFFLUENTS d a e ARE RECOVERED, RESPEC'HVELY, FROM WATER WASHING AND J.B.STARK VELUTION WITH (m -WATER. INvENToR BY Q ATTORNEY June 23, 1959 J.B. STARK SEPARATIONS OF NITROGENOUS ORGANIC COMPOUNDS Filed July 18,1955 .FIG.2

3 Sheets-Sheet 2 MODIFICATION B PuRINEs, PYRIMIDINES, NUCLEOSIDES ANDAMINO AcIDs b Co -WATER ELUANT SOLUTIONS APPLIED TO EXCHANGER SERIALLY,

ELUATES c AND d ARE, RESPECTlVELY,

THE FIRST PORTION OF THE ELUATE AND THE LATER PORTIO OF THE ELU TE,USING THE SAME E.I uANT IN EACH OAsE, i.e.,CO -WATER.

IN ORDERIo,b.

ANION E'XOHANGER 002 FORM 0 d FIRST ELUATE LATER ELUATE cONTAINs:cONTAINs: AM|NQ cms PURlN-ES, PYRIMIDINES AND NUCLEOSIDES.

J. B. STARK INVENTOR BY 3% ATTOR EY June 23, 1959 J. B. STARKSEPARATIONS OF NITROGENOUS ORGANIC COMPOUNDS Filed July 18, 1955 3Sheets-Sheet 3 MODIFICATION C cI b c MIXTURE To BE TREATED WASH v NH4OH- CONTAINS, FOR EXAMPLEI WATER WATER PURINES, PYRIMIDINES,'NUCLEO-ELUANT SIDES AND AMINO ACIDS I NEUTRAL ORGANIC COMPOUNDS,

e.g.,SUGARS.

cA IoN ExcHANGE IN H FORM SOLUTIONS APPLIED TO cATIoN EXCHANGERSERIALLY, IN ORDER o,b,c,

REMAINING ON cATIoN ExnHANGER AFTER ELIITIoN: INORGANIC cATIoNs, sucH AsI Nat d WASH WATER EFFLUE NT, CONTAINS:

SUGARS AND OTHER NEUTRAL ORGANIC COMPOUNDS..

INORGANIC ANIONS, SUCH AS ELUATE CONTAINS: PURINES, PYRIMIDINES,NUCLEOSIDES, AMINO ACIDS.

CHLORIDE SULPHATE efc..

NON-AMINO CARBOXYLIC ACIDS SUCH AS I AcTIc,GITRIc.

EFFLUENTS d AND e ARE RECOVE R E D, RESPECTIVELY, FROM WATER-WASHING ANDELUTION WITH NH' OH-WATER DE-AMMONIATED SOLUTION OF PURINES,PYRIMIDINES, NUCL-EOSIDES AND AMINO ACIDS.

MAY B FURTHER ELUATE TREATED TO EVAPORATE AMMONIA TREATED BY PRoc ss OFMODIFICATION A 0R PRocEss OF MODIFICATION B.

J.B. STAR K INVENTOR BY ATTORNEY United Stats SEPARATIONS OF NITR'OGENOUS ORGANIC COMPOUNDS John B. Stark, Berkeley, Calif., assignorto the United States of America as represented by the Secretary ofAgriculture A non-exclusive, irrevocable, royalty-free license in theinvention herein described, throughout the world for all purposes of theUnited States Government, with the power to grant sublicenses for suchpurposes, is hereby granted to the Government of the United States ofAmerica.

This invention relates to the isolation of chemical substances fromcompositions containing them. In particular, the invention has for itsprime object the separation of nitrogenous organic compounds such aspurines, pyrimidines, nucleosides, and amino acids from other substancesof an organic or inorganic nature. A specific object of the invention isthe provision of processes whereby purines, pyrimidines, nucleosides andamino acids are separated from source materials containing suchcompounds in admixture with non-amino carboxylic acids, inorganic salts,sugars and other impurities of an inorganic or organic nature. Otherobjects of the invention concern the provision of processes forpreparing the above-mentioned types of nitrogenous compounds in theirfree base form from salts thereof. Other objects of the inventionconcern the provision of novel procedures for utilizing and regeneratingion exchange materials. Further objects and advantages of the inventionwill be evident from the description herein taken in connection with theannexed drawing. I

In the drawing: Figs. 1, 2, and 3 are diagrammatic flow sheetsillustrating the procedures of modifications A, B, and C, respectively,in accordance with this invention.

Chemical compounds of the classes of purines and pyrimidines, also knownas purine bases and pyrimidine bases, occur in many biologicalmaterials, often combined with other substances to form nucleotides,nucleosides and nucleic acids. The purines and pyrimidines and theirderivatives play essential roles in the life cycle of animals andplants. It is often desired to isolate these compounds from thebiological substrates in which they occur for the purpose of conductingbiochemical investigations. In general, the separation of purines andpyrimidines by known procedures is cumbersome and inefficient. Aminoacids are likewise present in most plant and animal substances,particularly in combined form in proteinous materials. The separation ofamino acids from biological materials especially from hydrolysates ofproteinous materials is often desirable from the standpoint ofconducting biochemical investigations or to obtain the amino acid forindustrial purposes. Some of the amino acids such as glutamic acid,histidine, methionine, aspartic acid, lysine, etc. are used in thepreparation of flavoring agents, as feed supplements, nutritivepreparations, therapeutic agents; they are also useful as intermediatesin chemical synthesis, and so forth. In general, known methods ofseparating amino acids from biological sources are diificult andinefficient.

In accordance with this invention substances such as purine bases,pyrimidine bases, nucleosides, and amino acids can be separated frommixtures containing them or atom from one another by relatively simpleyet efficient processes. Basically, these processes involve noveltechniques of ion exchange. various modifications which are separatelydescribed hereinafter.

A basic feature of the invention involves a process wherein the mixtureis contacted with an anion exchange material followed by elution of theexchanger with an aqueous solution of carbon dioxide. By such atreatment, an effective separation is achieved in that the eluate willcontain for the most part the purines, pyrimidines, nucleosides, andamino acids originally present in the starting mixture. Remaining on theanion exchanger will be such compounds as non-amino carboxylic acids,inorganic acids, colored principles and so forth. Neutral organiccompounds, such as sugars, and inorganic cations will not be adsorbed bythe anion exchanger and will pass through the anion exchanger when theoriginal mixture is contacted therewith. They also can be removed fromthe exchanger prior to elution by washing with Water. The net result issummarized as follows:

(1) Present in the solution which passes through the anion exchanger orin the wash water: Sugars and other neutral organic compounds, inorganiccations such as Na K etc.

(2) Present in the CO -Water eluate: Purines, pyrimidines, nucleosides,and amino acids.

(3) Remaining adsorbed on the anion exchange material after elution WithCO -waterz Non-amino carboxylic acids, inorganic anions such aschloride, sulphate, etc., colored principles, etc.

The above explanation illustrates the results that are generallyachieved. However, in certain cases, depending on the chemical nature ofthe particular purines, pyrimidines, nucleosides, or amino acids,certain variations will occur. That is, some of these nitrogenouscomponents will be contained in the fractions other than the Co -watereluate. These exceptions are discussed and explained hereinafter. 7

It is a feature of the invention that the nitrogenous compounds areeluted with a solution of carbon dioxide in water. As a result theeluatecontains the desired nitrogenous compounds in aqueous solution.The carbon dioxide also present in the eluate can be readily removed byapplication of a moderate amount of heat leaving the desired compoundsin solution. Consequently, the isolation of the nitrogenous compounds isnot impeded by the presence of any undesired substance as would be thecase if the eluting solution contained a non-volatile compound or onewhich was volatile only with the application of a high degree oftemperature whereby the nitrogenous components would be exposed to thepossibility of decomposition.

In one modification of the invention, the anion exchanger material is inthe hydroxide form prior to application of the mixture. This procedure,in general, leads to separation of the purine bases, pyrimidine bases,nucleosides, and amino acids as a group from the associated impurities.This modification is explained in the part of this specificationentitled Modification A, and illustrated in Fig. l of the drawing.

In another modification, the basic process is varied in a certain way toachieve a difierent plan of fractionation. For example, if the anionexchanger is initially saturated with carbon dioxide, the mixture isapplied wit and the exchanger is then eluted with water saturated withcarbon dioxide, one can obtain a separation of amino acids from theother nitrogenous components (purines, pyrimidines, and nucleosides). Inthis modification, the amino acids will appear in the first runnings ofthe eluate whereas the other nitrogenous components will appear only inlater runnings of the eluate. This plan of opera- The inventionencompasses tion is explained at length below in the part of thisspecification entitled Modification B and illustrated in Fig. 2 of thedrawing.

The plan of fractionation may also be varied by apply ing cationexchanger treatment to the mixture before or after either of theaforesaid treatments with the anion exchanger. This mode of operation isexplained below in the part of this specification entitled ModificationC and illustrated in Fig. 3 of the drawing.

Another modification involves applying a preliminary treatment to theoriginal mixture, for example precipitation of the nitrogenous compoundsas their salts with an inorganic acid. The resulting salts may then besubjected to the anion exchange treatment to effectuate a furtherseparation and to produce the free bases. This procedure is explained inthe part of the specification entitled Modification D.

In operating under any of the modifications of the invention whereinelution with CO -Water is involved, one may employ conditions to securean eluting agent which contains more carbon dioxide than corresponds tosaturation at room temperature and atmospheric pressures. To this end,the eluate may be a refrigerated solution of CO in Water, saturated atthe refrigerated temperature. This temperature may be below roomtemperature and above the freezing point, for example, less than 20 C.and above C. At such temperatures the eluting solution will contain moreCO and consequently have greater power for eluting the desirednitrogenous compounds. As a result less of the eluting solution need beused and the eluate will be a more concentrated solution of the desirednitrogenous compounds. Instead of using a refrigerated CO -Watersolution (or, in addition to using such refrigerated solution) one mayconduct the elution under pressure. Thus the anion exchange material isheld in a pressure-resistant vessel and a CO -Water solution applied toit under superatmospheric pressure. Since the solubility of CO increaseswith pressure, it is obvious that by this means less water is needed andthe eluates contain the desired nitrogenous compounds in a moreconcentrated form. The degree of pressure is limited only by thepressure-resisting character of the equipment. In general, a range ofpressure from 1 to 200 lbs. per sq. in. gauge pressure in practicableand easily attainable with conventional pumping systems.

The invention is of particular value in the isolation of purines,pyrimidines, nucleosides, and amino acids from biological sourcematerials. The source material from which the nitrogenous compounds areisolated may be any one of extracts of plant or animal materials,hydrolysates of plant or animal tissue, and so forth. Examples ofstarting materials are liquids derived from such materials as animalpancreas, thymus, spleen, flesh, urine, guano, yeast, cofiee, tea,cocoa, and so forth. Particularly useful are waste liquors such as thoseobtained in the preparation of cane and beet sugars.

Examples of classes of compounds or individual compounds which may beisolated in accordance with this invention are:

Purine bases: adenine, guanine, hypoxanthine, caffeine, theobromine,paraxanthine, theophylline, 2-arninouric acid, 2,6-diaminopurine, etc.

Pyrimidine bases: uracil, thymine, cytosine, divicine, S-methylcytosine, 2-arnino-4-rnethyl pyrimidine, orotic acid, 6-methyluracil,6-aminouracil, 4,6-dihydroxypyrimidine, S-aminouracil, etc.

I Nucleosides: uridine, thymidine, adenosine, xanthosine, mosine,guanosine, cytidine, orotidine, S-methyl cytidine, etc.

Amino acids: glycine, alanine, valine, leucine, phenyl alanlne,tyrosine, tryptophane, lysine, arginine, methionine, histidine, glutamicacid, aspartic acid, and so forth.

Several modifications of the basic pattern, all within the scope of thisinvention are set forth below by Way of illustration but not limitation.

i MODIFICATION A In this process a solution containing at least one ofthe group purine bases, pyrimidine bases, nucleosides, and amino acidsand also various impurities is contacted with an anion exchanger in thebase form. The anion exchanger is then Washed with Water to removematerials not actually adsorbed on the exchanger but only mechanicallyheld in the mass. The wash water will contain such impurities as sugarsand other neutral organic compounds and inorganic cations such as Na K+and so forth. T he anion exchanger is then eluted with Water saturatedwith carbon dioxide. The eluate will contain the purine bases,pyrimidine bases, nucleosides, and amino acids. Remaining on theexchanger will be such impurities as non-amino carboxylic acids,inorganic anions, coiorcd principles, and the like. The non-aminocarboxylic acids can be recovered fromv the anion exchanger by elutionwith a strong acid such as aqueous hydrochloric acid. procedure of thismodification is depicted in the fiow sheet of Fig. l of the annexeddrawing.

A typical application of this procedure is illustrated in the followingexample:

Example A-l The starting material is a solution containing in 50 cc. ofWater the following ingredients:

Mg. Sucrose 10 Sodium chloride 1O Lactic acid (a non-amino carboxylicacid) l0 Hypoxanthine (a purine base) 5 Uracil (a pyrimidine base) 5Alanine (an amino acid) 10 The solution is poured onto a column of astronglybasic anion exchanger in the hydroxide form. The colum is Washedwith 50 cc. of water. Essentially all the sugar and sodium ions appearin the wash water passing through the column.

The column is then eluted with Water saturated at room temperature withcarbon dioxide. The elution is conducted continuously and the eluate iscollected in 10 cc. batches in separate test tubes. The tubes are testedfrom time to time to determine their contents. It is found that theeluate collected in tubes 20 to 30 inclusive contain essentially all theuracil, hypoxanthine, and alanine originally present and this eluatedoes not contain any lactic acid nor any chloride ions.

The lactic acid remaining on the column is then recovered by elutingwith 1 N hydrochloric acid.

The procedure outlined above can be generally applied to thosecompositions containing at least one member of the group: purine bases,pyrimidine bases, nucleosides, and amino acids in admixture with variousimpurities. Examples of specfic compounds within these groups are:

Purine bases: adenine, guanine, hypoxanthine, caffeine, theobromine,paraxanthine, 2,6-diaminopurine, etc.

Pyrimidine bases: cytosine, divicine, S-methyl cytosine,2-amino-4-methyl pyrimidine, uracil, thymine, 6-methyluracil,6-aminouracil, 4,6-dihydroxypyrimidine, S-aminouracil, etc.

Nucleosides: adenosine, xanthosine, inosine, guanosine, cytidine,uradine, thymidine, etc.

Amino acids: glycine, alanine, valine, leucine, phenyl alanine,tyrosine, tryptophane, lysine, arginine, histidine, etc.

As noted briefly above, in certain cases, depending on the nature of thenitrogenous components, the pattern of fractionation will vary somewhatfrom the general pattern. This situation is explained further below:

In cases where the original mixture contains an amino acid of the acidictype, that is, one in which the acidic properties outweigh the basicproperties, such amino acid will not be eluted by the CO -Water but willremain adsorbed on the anion exchanger. For example, if in the aboveexample (Example A--I) the original mixture contained glutamic acid,this acid would remain on the anion exchanger after the Co -waterelution and would appear together with the lactic acid in the finalelution with hydrochloric acid. Also included in this category of acidicamino acids is aspartic acid. The amino acids of the neutral or basictypes will not remain adsorbed on the anion exchanger but will be elutedby the carbon dioxide solution. Examples of such amino acids areglycine, alanine, valine, leucine, phenyl alanine, tyrosine,

tryptophane, lysine, arginine, histidine, etc.

In such instance where the acidic amino acid remains adsorbed on theanionexchanger, it can easily be separated by applying further ionexchange techniques. For instance, if in the procedure ofExample A-I,the original mixture contained glutamic acid this amino acid wouldremain on the anion exchanger after elution with CO water but wouldappear together with lactic acid in the final hydrochloric acid eluate.This eluate would then be contacted with a cation exchanger in thehydrogen form. Lactic acid would not be adsorbed by the cation exchangerbut would be removed by washing with water. Glutamic acid would beadsorbed by the cation exchanger and would be recovered therefrom byeluting with a solu- I tion of ammonium hydroxide.

Another exception is that if the original mixture has in it a purinecontaining a carboxyl group, a pyrimidine containing a carboxyl group,or the nucleosides of such purines or pyrimidines, then these will notbe eluted by the Co -water but will remain adsorbed on the anionexchanger. Such purines, pyrimidines, or their nucleosides can berecovered from the ion exchanger by eluting with a strong acid such ashydrochloric acid. Examples of compounds in this category are oroticacid and orotidine.

The fact that these nitrogeneous components of an acidic nature do notfollow the general pattern afiords a means by which these components maybe separated from the others, that is, those of a neutral to basicnature which follow the general pattern. Methods of doing so areillustrated below:

Example A- -ll The starting material is a solution containing, in 50 cc.of water, the following ingredients:

Mg. Sucrose Sodium chloride 10 Glutamic acid (an acidic amino acid) 10Alanine (a neutral amino acid) 10 The starting material is a solutioncontaining in 50 cc. of water the following ingredients:

Mg. Orotic acid (a carboxylic pyrimidine) 5 Uracil (a non-carboxylicpyrimidine) 5 The solution is poured onto a column of a stronglybasicanion exchanger in the hydroxide form. The col umn is eluted with about200 cc. of water saturated with carbon dioxide whereby uracil isobtained in the eluate. The column is then washed with 1 N hydrochloricacid whereby orotic acid is obtained in this eluate.

MODIFICATION B V In this modification pt the invention, the mixture tobe resolved is contacted with an anion exchanger which has beenpreviously saturated with carbon dioxide. That is, water saturated withcarbon dioxide is previously run through the anion exchanger until itwill absorb no more carbon dioxide. The mixture is then put on the anionexchanger and the exchanger is then eluted with water saturated withcarbon dioxide. It has been observed that by such technique the aminoacids can be separated from the other nitrogenous components (purinebases, pyrimidine bases andvnucleosides) because the amino acids areeluted before these other components. Thus the first portions of theeluate contain the amino acids whereas the purine bases, pyrimidinebases, and nucleosides will appear only in later portions of the eluate.

A distinct advantage of this plane of operation is that the elutionautomatically regenerates the anion exchanger and puts it into conditionfor treatment of the next batch of material. No separate regenerationstep is needed therefore.

Preferably the procedure of this modification is applied to a materialwhich has already been partly purified, for example, by the anionexchanger treatment described in Modification A or the cation exchangetreatment described in Modification C. The point is that a water washingtreatment cannot successfully be used in this technique and as a resultif the mixture applied to the anion exchanger contains impurities suchas sugars, inorganic salts, etc., these impurities will appear in theeluate with the desired nitrogenous components. It is therefore thepreferred practice to first remove these impurities by the aniontreatment of Modification A, by the cation treatment of Modification C,or by other means. This point .is further illustrated as follows:

Where the process of this modification is applied to the mixturecontaining various impurities such as sugars, inorganic salts, inorganicacids, non-amino carboxylic acids, salts of non-amino carboxylic acids,etc., the separation achieved will be as tabulated below:

(1) Present in the CO -Water eluate: (a) The first portions of theeluate will contain the sugars and other neutral organic compounds;salts of non-amino carboxylic acids, such as sodium lactate, potassiumcitrate, etc.; inorganic salts such as NaCl, KCl, etc.; and the aminoacids. (b) The subsequent portions of the eluate will conltain thepurine bases, pyrimidine bases, and nucleosr es.

(2) Remaining on the column after elution with CO water: Non-aminocarboxylic acids, such as formic, lactic, acetic, oxalic, succinic,etc.'and inorganic acids such as hydrochloric, sulphuric, phosphoric,etc.

It is to be noted that in this mode of operation where the mixturecontains salts of inorganic acids or salts of non-amino carboxylicacids, these compounds will pass through the anion exchanger and willappear as such in the first portions of the eluate. On the otherhand,where the mixture contains inorganic acids or non-amino carboxylicacids, present as such acids, rather than as salts, these acids will beadsorbed on the column and will remain on it even after elution with theCo -water solution.

In the event that the original mixture is first at least partiallypurified so that it contains essentially only purine bases, pyrimidinebases, nucleosides, and amino acids, a separation is achieved asfollows:

(1) Present in cO -water eluate: The first portions of the eluate willcontain the amino acids; the subsequent portions of the eluate willcontain the purine bases, pyrimidine bases, and nucleosides.

(2) Remaining on the anion exchanger: Nothing. The eluted exchanger isthus completely cleared and regenerated and is ready for treatment ofthe next batch of material.

The procedure of this modification is depicted in the flow sheet of Fig.2 of the annexed drawing.

The starting material is a solution containing, in 50 cc. of water, thefollowing ingredients:

Mg. Adenine 10 Alanine 10 The solution is poured onto a column of astronglybasic anion exchanger previously treated to saturation withaqueous carboii dioxide. The column is eluted with water saturated withcarbon dioxide. The eflluent is ,collected in 10 cc. batches in separatetubes. It is observed that the alanine is present in tubes 4 to 10, theconcentration of alanine being at a maximum in tube 6. The adenine ispresent in tubes 20 to 80, the concentration of adenine being at amaximum in tube 40. After this elution is complete, the column is readyfor separation of another lot of purine-amino acid or similar mixture.

The procedure described and illustrated above may be generally appliedto separate amino acids from their admixture with components of thegroup-purine bases, purimidine bases, and nucleosides. Examples ofspecific compounds in these groups are:

Purine bases: adenine, guanine, hypoxanthine, caffeine, theobromine,paraxanthine, theophylline, Z-amiiiouric acid, 2,6-diaminopurine, etc.

Purimidine bases: cytosine, divicine, S-methyl cytosine,2-amino-4-methyl pyrimidine, uracil, thymine, 6-methyl uracil,S-aminouracil, 4,6-dihydroxypyrimidine, S-aminouracil, etc.

Nucleosides: adenine, xanthosine, inosine,. guanosine, cytidine,uridine, thymidine, etc.

Amino acids: glycine, alanine, valine, leucine, phenyl alanine,tyrosine, tryptophane, lysine, arginine, histidine, etc.

In certain cases the pattern of fractionation will not follow thegeneral pattern. Thus in this procedure, where the mixture being treatedcontains an acidic amino acid (such as glutamic or aspartic) such aminoacid will not be eluted by the CO -water but will remain adsorbed on theanion exchange. Such compounds can subsequently be recovered by elutionwith a solution of a strong acid such as hydrochloric. Likewise purinebases or pyrimidine bases which contain carboxyl groups or thenucleosides of these will not be eluted by the CO -water but will remainadsorbed on the anion exchanger. Such compounds, examples of which areorotic acid orotidine, can subsequently be recovered by elution with astrong acid such as hydrochloric.

The exceptional behavior of the nitrogenous components of an acidicnature can be utilized as a tool to effect separation of such componentsfrom others which follow the general pattern, that is, those which havea neutral to basic nature. This technique is illustrated below.

Example B-II The starting material is a solution containing in 50 cc. ofwater, the following ingredients:

Mg. Aspartic acid (an acidic amino acid) Glycine (a neutral amino acid)5 Adenine 5 MODIFICATION C In this process, the initial mixture issubjected to treat ment with a cation exchanger. The resulting purifiedmaterial may then be subjected to treatment with an anion exchangerusing the plan of Modification A or B. Thus the anion exchanger may bein the 'hY form (Modification A-) or in the carbon-dioxid Saturated form(Modification B).

'Ilhe treatment with the cation exchanger has the advantage that variousimpurities are removed from the mixture, such impurities includinginorganic salts, nonamino carboxylic acids and neutral organic compoundssuch as sugars.

The mixture -to be treated in accordance with this modificationmaycontain any of the purine bases, pyrimidine bases, nucleosides, oramino acids. together with various .impurities such as non-aminocarboxylic acids, neutral organic compounds, inorganic salts, coloredprinciples, and so forth.

Application of the .process of this modification entails firstcontacting the mixture with a cation exchanger in the hydrogen form. Thecation exchanger is then washed with water to remove materials notactually adsorbed on the exchanger but only mechanically held in themass. The wash water will contain such impurities as inorganic anions(chloride, sulphate, etc.); non-amino carboxylic acids (lactiqoxalic,succinic, citric, etc.); and neutral organic compounds as for examplesugars. The cation exchanger'is then eluted with an aqueous solution ofammonium hydroxide. The eluate contains the purine bases, pyrimidinebases, nucleosides, and amino acids. Remaining adsorbed on the cationexchanger are such impurities asinorganic cations (potassium, sodium,etc.) and colored principles.

The ammonium hydroxide eluate is then heated to evaporate'the ammoniatherefrom. The resulting solution may then be treated with an anionexchanger by the previously described methods.

'llhe net result of the cation exchange treatment is as follows:

(1) Present in the solution which passes through the cation exdhanger'orpresent in the wash water: Sugars and other neutral organic compounds;chlorides, sulphates, or other inorganic anions-and non-amino carboxylicacids such as formic, acetic, succinic, oxalic, lactic, citric, etc. asmay be present in the original mixture. If large amounts of inorganicsalts (KCl, NaCl. etc.) are present in the original mixture these willappear as such in the wash water.

(2) Present in the ammonium hydroxide eluate: Purine bases,pyrimidine-bases, nucleosides, and amino acids.

(3) Remaining adsorbed on the cation exchanger after elution withammonium hydroxide solution: Inorganic cations such as sodium andpotassium, colored principles, and so forth.

The procedure of this modification is further illustrated in the flowsheet diagram of Fig. 3 of the annexed drawing.

It is a feature of the cation exchange treatment that the eluting agentcontain only water and ammonium hy droxide. As a result the desirednitrogenous compounds are obtained in an eluate containing ammoniumhydroxide as the only contaminant. This ammonium hydroxide can bereadily removed by application of a moderate amount of heat leaving thedesired compounds in solution. Consequently, the isolation of thenitrogenous compounds is not impeded by the presence of any undesiredcompound as would be the case if the eluting solution contained anon-volatile material or one volatile only by the application of anamount of high degree of temperature in which case the nitrogenouscompounds would be exposed to danger of decomposition.

The above explanation illustrates the results that are generallyachieved. However in certain cases, depending on the chemical nature ofthe particular purines, pyrimidines, nucleosides, or amino acids,certain variations will occur. That is, some of these nitrogenouscomponents 9. will be contained in the fractions. other than theammonium hydroxide eluate. Theseexceptions are discussed and explainedhereinafter.

The following example illustrates typical application of the plan ofthis modification-of the invention.

The startingmaterial is a solution containing, in 50 cc. water, thefollowing ingredients;

The solution is poured onto a column of a stronglyacidic cationexchanger in the hydrogen form. The column is washed with Water.Essentially all the sugar, lactic acid, and the chloride ions appear inthe wash water.

The column is then eluted with 2% aqueous ammonium hydroxide solution.The eluate contains essentially all of the hypoxanthine, glutamic acid,and alanine together with a minor amount of potassium ions. Most of thepotassium ions'remain adsorbed on the cation exchanger.

The ammonium, hydroxide eluate is warmed on a steam bath until theammonia is evaporated. The resulting solution of hypoxanthine, glutamicacid and alanine is re solved as follows:

The solution is poured onto a column of a strongly-basic anion exchangerwhich has been previously saturated with aqueous carbon dioxide. Thecolumn is eluted with water saturated with carbon dioxide. The firstrunnings oi the eluate contain all the alanine; later runnings of eluatecontain all the hypoxan'thine. The glutamic acid remains on the anionexchanger and may be eluted with aqueous hydrochloric acid. Y I v Theprocedure described above may be generally applied to those compositionscontaining one or more members of the group'purine bases, pyrimidinebases, nuc1e osides, and amino acids in admixture with variousimpurities. Examples of specific'co rnpound's within these groups are:

Purine bases: adenine, guanine, hypoxanthine,cafieine, theobromine,paraxanthine, theophylline, 2-aminouric acid, 2,6-diaminopurine, etc.

Pyrimidine bases: cytosine, divicine, 5-methyl cytosine,2-amino-4-methyl pyrimidine, 6-aminouracil, S-aminouracil, etc.

Nucleosides: adenine, xanthosine, inosine, guanosine, cytidine, etc.

Amino acids: glycine, alanine, valine,-leucine, phenyl alanine,tyrosine, tryptophane, lysine, arginine, histidine, glutamic acid,aspartic acid, etc.

As noted briefly above, in certain cases, depending on the nature of thenitrogenous components, the pattern of fractionation in the cationexchange treatment will vary somewhat from the general pattern. Thissituation is explained as follows: 1

In cases Where the original mixture contains a purine containing acarboxyl group, a pyrimidine containing a carboxyl group, or theirrespective nucleosides, these compounds Will not be adsorbed by thecation exchanger but will appear in the water efiluent when the cationexchanger is washed with water. Included in this category are suchcompounds as orotic acid and orotidine. For instance, if'in the aboveexample (Example C-I) the original mixture contained orotic acid, thiscompound would appear in the wash water effluent together with sugar,lactic acid, and chloride ions. Also included in this category ofcompounds which are not absorbed by the cation exchanger are purines,pyrimidines, or their respective nucleosides which exhibit acidicproperties even though they do not contain carboxyl groups. Spec'ific'examples 'of su'ch' compounds are uracil, uridine, thymine, thymidine,4,6-dihydroxypyrimidine and 6- me y fl- I The exceptionsnoted' above maybe utilized for the separation of the compounds of an acidic nature fromthe compounds which follow the general pattern, that is, those ofa'neutral to basic nature. Such .a procedure is illustrated below:

The starting material is a solution containing in 50 cc. of water, thefollowing ingredients:

. t Example C-II Mg. Inosine 5 Guanosine 5 Adenosine 5 Xanthosine' 5Uridine- 5 The solution is'poured onto a column of a stronglyaci diccation exchanger in the hydrogen form. The column is then washed withwater. The aqueous efiluent contains essentially all theuridine. Thecolumn is then eluted with. 2% aqueous ammonium hydroxide; the eluatecontains the other nucleosides (isosine, guanosine, adenoside, andxanthosine);

' Example C-JII 1000v grams of beet molasses is agitated with 1000gramsof activated carbon. This mixture is washed with water to. removemost of the sugar, then extracted with concentrated ammonium hydroxide(40 liters) to obtain an extract containing various nitrogenous basesoriginally present inthe molasses. The solution is heated on the steambath toevaporate' the ammonia therefrom.

The resulting solution, (5 liters) is poured onto a column of astrongly-acidic cation exchanger in the hydrogen form; The exchanger isyvashed with water. The wash water effluent is collected. It containsuracil and uridine in addition to other nitrogenous bases and otherimpurities. i

This efiluent is then poured onto a column of a stronglybasic anionexchanger in the hydroxide form. The column is washed with? water-theneluted with aqueous carbon dioxide solution. The eluate is foundtocontain uracil and uridine.

MODIFICATION D Another modification of the invention is concerned withthe conversion of the saltsof purine bases, pyrimidinebasesfnucleosides, or amino acids into the corresponding free bases.illustrative examples are the conversion ofaden'ine hydrochloride oradenine sulfate into free adenine, the conversion of glycinehydrochloride or sulphate into free glycine, and vso forth.

Such a conversion-from the salt form into the free base may be anincident ,toa purification treatment, for instance, where thenitrogenouscompounds are precipitated as the hydrochlorides byconcentration of a bio logical source solution followed by addition ofhydrochloric acid. Previous methods for preparing the free bases fr'omtheirisalts are in general cumbersome and unsatisfactory. i

The procedure in accordance with this invention involv'es adsorbingthesalt on an anion exchanger in the hydroxideform and then selectivelyeluting the base with an aqueous solution of carbon dioxide. The netefifect of the adsorption and elution is that'a separation is made ofthe original cation and anion moieties of the original salt. 7 Theeluate maybe subjected .to concentration to obtain the solid 'free baseorit may be treated with another acid to obtain a salt difierent fromthe original one.

'A' feature or; the invention isthat the eluate containing the freebases contains only volatile materials-Water and CO Both of these areeasily removed by evaporation without decomposition of the base. Therecovery of the free base is not impeded by the presence of non-volatilecomponents as is the case in prior art methods. Another advantage isthat neither the salt nor the free base come into contact with strongacids or alkalis and thus the possibility of decomposition by hydrolysisor the like is completely avoided. A further advantage is that if thereis an excess of acid admixed with the salt, this excess acid togetherwith the portion of the acid derived from the salt will be adsorbed onthe exchanger. For example, if a mixture of alanine hydrochloride andhydrochloric acid is subjected to the process of this modification,alanine will be obtained in the CO -Water eluate whereas remaining onthe exchanger will be the hydrochloric acid derived both from the saltand from the excess acid admixed with the salt. The same propositionholds where the excess acid is different from the acid combined in thesalt, e.g., where the initial mixture contains alanine hydrochloride andsulphuric acid; alanine sulphate and hydrochloric acid; or other salt ofa relatively strong acid with a purine, pyrimidine, nucleoside or aminoacid together with an excess of such acid.

This modification is further illustrated as follows:

Example DI Ten milligrams of the hydrochloride of 2-amino4methyl-pyrimidine is dissolved in 1 cc. of water. This solution ispoured onto a column containing 10 cc. of a strongly basic anionexchanger in the hydroxide form. The column is then eluted with watersaturated with carbon dioxide. The resulting eluate containing the freebase 2amino4-methyl pyrimidine is evaporated on a steam bath to recoverthe base in solid form.

Example D-II The procedure as described in Example DI is repeated usingdifferent salts under the same conditions. The salts used are:

Adenine hydrochloride Tyrosine hydrochloride Alanine hydrochlorideLysine hydrochloride In each case the free base is obtained in the CO-Water eluate.

The procedure disclosed above can be generally applied to produce thefree base form of purine bases, pyrimidine bases, nucleosides, and aminoacids from their salts with i.

acids which are substantially stronger than carbonic acid. Thus the freebases may be produced from salts in which the bases are combined withsuch acids, as for example, acetic, benzoic, chloroacetic, citric,dichloroacetic, formic, lactic, maleic, oxalic, hydrobromic, phosphoric,sulphuric, picric, sulphurous, and so forth. The base (cation) portionor th salts may be any of the purine bases, pyrimidine bases,nucleosides or amino acids which are described herein as following thegeneral pattern of isolation by the techniques of Modification A or B.Illustrative samples are:

Purine bases: adenine, guanine, hypoxanthine, caffeine, theobromine,paraxanthine, theophylline, Z-aminouric acid, 2,6-diaminopurine, etc.

Pyrimidine bases: cytosine, divicine, S-methyl cytosine,2-amino-4-methyl pyrimidine, 2,4,6-triaminopyrimidine,2,4,5,6-tetraamino pyrimidine, 4-amino-2,6-dihydroxypyrimidine, etc.

Nucleosides: adenosine, xanthosine, inosine, guanosine, cytidine,uridine, thymidine, etc.

Amino acids: glycine, alanine, valine, leucine, phenyl alanine,tyrosine, tryptophane, lysine, arginine, histidine, etc.

The process of this modification does not operate in the general mannerwith salts of acidic amino acids, such as glutamic or aspartic acids, asthese compounds will not be eluted by the CO -Water solution asexplained hereinabove in connection with Modification A. However, theprocedure may be applied to mixtures of salts of two or more amino acidsin which case one will attain the dual result of (1) preparation of thefree bases from the salts and (2) separation of the components into twofractionsthe ones of a neutral to basic nature which are eluted by theCo -water solution and the others of an acidic nature which remainadsorbed on the anion exchanger. Such a procedure is illustrated in theexample below. It is observed that purine bases, pyrimidine bases andnucleosides which exhibit acidic properties do not form salts withacids. Examples of such compounds are uracil, uridine, thymine,thymidine, orotic acid, orotidine, 4,6-dihydroxy pyrimidine, etc. Ifthese compounds are present 11 the mixture being subjected to thetreatment of this modification, those with carboxyl groups (orotic acid,orotidine) will remain adsorbed on the exchanger and will not be elutedby the CO -water solution; those which are of an acidic nature but whichdo not have carboxyl groups (uracil, thymine, 4,6-dihydroxypyrimidine,uridine, thymidine, etc.) will be eluted with the CO -water solution andthus appear in the eluate.

Example DIII The starting material is a solution containing, in 50 cc.of water, the following ingredients:

The solution is poured onto a column of a stronglybasic anion exchangerin the hydroxide form. The exchanger is then eluted with water saturatedwith carbon dioxide. The eluate contains free adenine. The glutamic acidcan be recovered from the exchanger by elution with hydrochloric acid.There is thus achieved the preparation of adenine as the free base andits separation from the glutamic acid.

It is to be noted that in the procedure of this modification the anionexchanger is initially in the hydroxide form and the exchanger may bewashed with water after placing the salt or salts on it but prior toelution with the CO -water. By such washing, impurities not adsorbed bythe anion exchanger may be removed so that a purification will thus beachieved. The impurities so removable by washing may be for examplesugar and other neutral organic compounds, inorganic cations, and soforth.

Having thus described the invention, what is claimed l. The processwhich comprises applying to a stronglybasic anion exchange material animpure solution containing a purine base, thereafter eluting the anionexchange material with an aqueous solution of carbon dioxide andcollecting the eluate being a purified solution of the purine base.

2. The process which comprises applying to a stronglybasic anionexchange material a solution of biological origin containing a purinebase, the solution also containing acidic impurities and neutralimpurities, water-washing the anion exchange material, then eluting itwith an aqueous solution of carbon dioxide and collecting the eluatebeing a solution containing the purine base, said eluate beingessentially free from the acidic and neutral impurities present in theoriginal solution.

3. A process for separating amino acids from a composition containingamino acids in admixture with at least one other nitrogenous compound ofthe group consisting of purine bases, pyrimidine bases and nucleosideswhich comprises applying said composition to an anion exchangersaturated with carbon dioxide, eluting the exchanger with an aqueoussolution of carbon dioxide and recovering a first portion of the eluatecontaining the amino acids and a second portion of the eluate containingsaid other nitrogen compound.

4. A process for separating an amino acid from a composition containingan amino acid and a purine base which comprises applying saidcomposition to an anion exchanger saturated with carbon dioxide, elutingthe exchanger with an aqueous solution of carbon dioxide, and recoveringa first portion of the eluate containing the amino acid and a secondportion of the amino acid con taining the purine base.

5. A process for separating an amino acid from a composition containingan amino acid and a pyrimidine base which comprises applying saidcomposition to an anion exchanger saturated with carbon dioxide, elutingthe exchanger with an aqueous solution of carbon dioxide, and recoveringa first portion of the eluate containing the amino acid and a secondportion of the eluate containing the pyrimidine base.

6. A process for separating an amino acid from a composition containingan amino acid and a nucleoside which comprises applying the compositionto an anion exchanger saturated with carbon dioxide, eluting theexchanger with an aqueous solution of carbon dioxide, and recovering afirst portion of the eluate containing the amino acid and a secondportion of the eluate containing the nucleoside.

7. A process of fractionating a composition containing at least onemember of the group consisting of purine bases, pyrimidine bases,nucleosides and amino acids, said composition containing at least one ofsuch compounds of a neutral to preponderantly basic nature, saidcomposition also containing at least one of said compounds of apreponderantly acidic nature, which comprises adsorbing said compositionon a strongly-basic anion exchanger, eluting the exchanger with anaqueous solution of carbon dioxide, recovering the eluate containingessentially only the components of a neutral to preponderantly basicnature, the components of a preponderantly acidic nature remainingadsorbed on the exchanger, then eluting the latter components with astrong acid.

8. A process of fractionating a composition containing at least onemember of the group consisting of purine bases, pyrimidine bases,nucleosides, and amino acids, said composition including at least one ofsuch compounds of a preponderantly acidic nature and at least one purinebase of a neutral to preponderantly basic nature, which comprisesadsorbing said composition on a strongly-basic anion exchanger, elutingthe exchanger with an aqueous solution of carbon dioxide, recovering theeluate containing essentially only the said purine base of a neutral topreponderantly basic nature, the components of a preponderantly acidicnature remaining adsorbed on the exchanger, and eluting the latter witha strong acid.

9. A process of fractionating a composition containing at least onemember of the group consisting of purine bases, pyrimidine bases,nucleosides, and amino acids, said composition including at least one ofsuch compounds of a preponderantly acidic nature and at least onepyrimidine base of a neutral to preponderantly basic nature, whichcomprises adsorbing said composition on a strongly-basic anionexchanger, eluting the exchanger with an aqueous solution of carbondioxide, recovering the eluate containing essentially only the saidpyrimidine base of a neutral to preponderantly basic nature, thecomponents of a preponderantly acidic nature remaining adsorbed on theexchanger, and eluting the latter with a strong acid.

10. A process of fractionating a composition containing at least onemember of the group consisting of purine bases, pyrimidine bases,nucleosides, and amino acids, said composition including at least one ofsuch compounds of a preponderantly acidic nature and at least onenucleoside of a neutral to preponderantly basic nature, which comprisesadsorbing said composition on a. strongly-basic anion exchanger, elutingthe exchanger with an aqueous solution of carbon dioxide, recovering theeluate containing essentially only the said nucleoside of a neutral topreponderantly basic nature, the components of a preponderantly acidicnature remaining adsorbed on the exchanger, and eluting the latter witha strong acid.

11. A process of fractionating a composition contain ing at least onemember of the group consisting of purine bases, pyrimidine bases,nucleosides, and amino acids, said composition including at least one ofsuch compounds of a preponderantly acidic nature and at least one aminoacid of a neutral to preponderantly basic nature, which comprisesadsorbing said composition on a strongly-basic anion exchanger, elutingthe exchanger with an aqueous solution of carbon dioxide, recovering theeluate containing essentially only the said amino acid of a neutral topreponderantly basic nature, the components of a preponderantly acidicnature remaining adsorbed on the exchanger, and eluting the latter witha strong acid.

References Cited in the file of this patent Winters et al.: Ind. andEng. Chem, March 1949, pages 460-463.

Cohn: Science, volume 109 (1949), pages 377-78.

Nachod: Ion Exchange, published by Academic Press (New York), 1949,pages 151 to 152 relied on.

Kunin et al.: Ion Exchange Resins, published by Wiley & Sons (New York),1950, pages 41, 118 to 120, and 132 to 135.

Kunin et al.: Industrial and Engineering Chemistry, volume 45 (1953),page 84.

Kunin et al.: Industrial and Enginee Chemistry volume 46 1954 page 119.mg

7. A PROCESS OF FRACTIONATING A COMPOSITION CONTAINING AT LEAST ONEMEMBER OF THE CONSISTING OF PURINE BASES, PYRIMIDINE BASES, NUCLEOSIDESAND AMINO ACIDS, SAID COMPOSITION CONTAINING AT LEAST ONE OF SUCHCOMPOUNDS OF A NEUTRAL TO PREPONDERANTLY BASIC NATURE, SAID COMPOSITIONALSO CONTAINING AT LEAST ONE SAID COMPOUNDS OF A PREPONDERANTLY ACIDICNATURE, WHICH COMPRISES ADSORBING SAID COMPOSITION ON A STRONGLY-BASICANION EXCHANGER, ELUTING THE EXCHANGER WITH AN AQUEOUS SOLUTION OFCARBON DIOXIDE, RECOVERING THE ELUATE CONTAINING ESSENTIALLY ONLY THECOMPONENTS OF A NEUTRAL TO PREPONDERANTLY BASIC NATURE, THE COMPONENTSOF A PREPONDERANTLY ACIDIC NATURE REMAINING ADSORBED ON THE EXCHANGER,THEN ELUTING THE LATTER COMPONENTS WITH A STRRONG ACID.